Transcriptional landscape of the embryonic chicken Müllerian duct

Abstract

Background: Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract in vertebrates. Many disorders of female reproduction can be attributed to anomalies of Müllerian duct development. However, the molecular genetics of Müllerian duct formation is poorly understood and most disorders of duct development have unknown etiology. In this study, we describe for the first time the transcriptional landscape of the embryonic Müllerian duct, using the chicken embryo as a model system. RNA sequencing was conducted at 1 day intervals during duct formation to identify developmentally-regulated genes, validated by in situ hybridization.

Results: This analysis detected hundreds of genes specifically up-regulated during duct morphogenesis. Gene ontology and pathway analysis revealed enrichment for developmental pathways associated with cell adhesion, cell migration and proliferation, ERK and WNT signaling, and, interestingly, axonal guidance. The latter included factors linked to neuronal cell migration or axonal outgrowth, such as Ephrin B2, netrin receptor, SLIT1 and class A semaphorins. A number of transcriptional modules were identified that centred around key hub genes specifying matrix-associated signaling factors; SPOCK1, HTRA3 and ADGRD1. Several novel regulators of the WNT and TFG-β signaling pathway were identified in Müllerian ducts, including APCDD1 and DKK1, BMP3 and TGFBI. A number of novel transcription factors were also identified, including OSR1, FOXE1, PRICKLE1, TSHZ3 and SMARCA2. In addition, over 100 long non-coding RNAs (lncRNAs) were expressed during duct formation.

Conclusions: This study provides a rich resource of new candidate genes for Müllerian duct development and its disorders. It also sheds light on the molecular pathways engaged during tubulogenesis, a fundamental process in embryonic development.

Keywords: Chicken embryo; FOXE1; Müllerian duct; OSR1; RNA-seq; Sex determination.

Fig. 1

Schematic figure of chicken embryo Müllerian duct tissue sampling and pipeline of the bioinformatical analysis. a Samples were taken on 3 consecutive days, E4.5, E5.5 and E6.5. Posterior mesonephros at day 4.5 was used as control (red square). The 4.5 anterior sample comprised both duct and mesonephric kidney tissue. Pure Müllerian duct tissue was collected only at E5.5 and E6.5. b Pipeline of RNA-seq bioinformatical analysis. For static comparisons, differentially expressed genes (DEGs) were identifed by comparing each stage to the control tissue (E4.5 posterior mesonephros) (Green arrows). For dynamic comparisons, DEG’s were identified by comparing successive stages of duct development (blue arrows). The datasets were subjected to a number of bioionformatic analyses, including GO terms, PPI, WCGNA and TF developmental clustering. c Principle component analysis (PCA) analysis of all samples using all genes. d Differentially Expressed Genes (DEG’s) across samples. Up-regluated genes shown in red, down-regulated genes shown in blue. An increasing number of duct genes were up-regulated relative to the control (E4.5 posteroir) as duct development proceeded. e Venn diagrams showing shared DEGs based on static comparisons (E4.5 anterior, E5.5 and E6.5 duct comparsed to control tissue, E4.5 posterior mesonehpric kidney.) 906 genes were differentially expressed in all samples relative to the control. f Venn diagrams showing shared DEGs based on dynamic comparisons (comparing successive stages of duct development). One hundred eight genes that showed differential expression across all stages of duct development

Fig. 2

Gene Ontology analysis of DEGs, based on static and dynamic comparisons. a Bar plots showing gene ontology-based number of DEG enrichment map analysis to identify enriched pathways in static comparisons (E4.5 Anterior, E5.5 and E6.5 each compared to the control tissue (E4.5 Posterior mesonephros. b Bar plots showing gene ontology-based number of DEGs enrichment map analysis to identify enriched pathways in dynamic comparisons (E4.5 Anterior vs E5.5 and E5.5 duct E6.5 each compared to the control tissue (E4.5 Posterior mesonephros

Fig. 3

Gene networks of the top enriched modules at each stage of duct formation. Networks were visualized using top 50 or 100 highly connected genes. Colour of nodes show their DEG status. Pink nodes are statically DEGs (compared to stage 4.5 posterior control) and red nodes are DEGs in both static and dynamic comparisons. For dynamic changes it has been compared with the previous stages except for E4.5-Ant. (compared to 4.5 posterior stage). Grey nodes are non-DEGs. Larger nodes have higher degree of “hubness”. Only 500 or 1000 highly weighted edges are shown. Thicker edges (darker colour) represent higher weight

Fig. 4

Heatmap plot showing clustering of all transcription factors expressed during chicken Mullerian duct development and GO of their members. Different colours are related to each cluster. Key of the heatmap is after scaling the CPM; red = highly expressed, blue = lowly expressed. Numbers beside each bar represent P-values

Fig. 5

Expression of top genes differentially expressed during embryonic Müllerian duct formation (2 > log2 FC; with cpm > 50 by E6.5). a Expression from the RNA-seq data for the top five DE genes, enriched over duct development (cpm). b RT-PCR analysis of gene expression in isolated ducts over E5.5–6.5. Detection of mRNA transcripts of COL1A2, SMARCA2, POSTN, PRICKLE, TSHZ3, HTRA3, TGFBI, RUNX1, FOXE1, LOXL2 and OSR1. (RT- = no RT enzyme; WE = whole embryo at E4.5). Figure shows cropped gel images for clarity. (Un-cropped gel images shown in Supplementary Figure 6)

Fig. 6

Whole mount in situ hybridization and sectioned whole mounts for candidate duct genes, in E6 (stage 29) urogenital systems (UGS). a COLA2, widely expressed in the UGS and in duct mesenchyme (MDM). b POSTN, localised in the mesenchyme. c, TGFBI, strongly expressed in the Müllerian duct epithelium (MDE). d SMARCA2, strongly expressed in the duct mesenchyme (MDM). e FOXE1, expressed in the duct mesenchyme (MDM). f OSR1, expressed in duct mesenchyme (MDM). g PRICKLE, expressed in the duct epithelium (MDE). h. RUNX1, strongly expressed in the duct epithelium (MDE) and also in the mesenchyme (MDM)

Fig. 7

Expression profile of FOXE1 mRNA in the embryonic chicken Müllerian duct. a Time course of FOXE1 mRNA expression in the male and female Müllerian duct during development, as assayed by whole mount in situ hybridization and sectioned whole mounts. Expression is first detected in female and male Müllerian duct at E5.5 (stage 28) and 6.5 (stage 30). At E8.5 (stage 34), FOXE1 is strongly expressed in the developing female ducts, but weak staining is detected in male ducts. Expression is not detected in gonads and mesonephros. Sectioned whole mounts (upper images) show expression of FOXE1 detectable throughout the developmental stages in Müllerian duct mesenchyme (MDM), with no expression in Müllerian duct epithelium (MDE). At E8.5 (stage 34), FOXE1 is strongly expressed in the developing female ducts but weak expression in male ducts. b Quantitative RT-PCR analysis of FOXE1 mRNA expression in E5.5, 6.5 and E8.5 male and female Müllerian ducts. Expression levels were normalized to β-ACTIN and expressed relative to E5.5. Bars represent Mean ± SEM. ** = adjusted p value< 0.01. One-way ANOVA and Tukey’s multiple comparisons test

Fig. 8

Expression of lncRNAs in chicken Müllerian ducts. Top differentially expressed lncRNAs are shown. a RNA-seq data, showing increasing expression of three uncharacterised lncRNAs during duct formation. b In situ hybridization analysis of lncRNA LOC107052410.This non-coding RNA shows polarised expression E6.0 (stage 29) Müllerian ducts, localised to the duct mesenchyme. (MDM)